Thelytokous Parthenogenesis in the Fungus-Gardening
Ant Mycocepurus smithii (Hymenoptera: Formicidae)
Christian Rabeling1*, José Lino-Neto2, Simone C. Cappellari1, Iracenir A. Dos-Santos2, Ulrich G. Mueller1,
Maurı́cio Bacci, Jr.3
1 Section of Integrative Biology, The University of Texas at Austin, Austin, Texas, United States of America, 2 Departamento de Biologia Geral, Universidade Federal de
Viçosa, Viçosa, Minas Gerais, Brazil, 3 Center for the Study of Social Insects, São Paulo State University, Rio Claro, São Paulo, Brazil
Abstract
The general prevalence of sexual reproduction over asexual reproduction among organisms testifies to the evolutionary
benefits of recombination, such as accelerated adaptation to changing environments and elimination of deleterious
mutations. Documented instances of asexual reproduction in groups otherwise dominated by sexual reproduction
challenge evolutionary biologists to understand the special circumstances that might confer an advantage to asexual
reproductive strategies. Here we report one such instance of asexual reproduction in the ants. We present evidence for
obligate thelytoky in the asexual fungus-gardening ant, Mycocepurus smithii, in which queens produce female offspring
from unfertilized eggs, workers are sterile, and males appear to be completely absent. Obligate thelytoky is implicated by
reproductive physiology of queens, lack of males, absence of mating behavior, and natural history observations. An obligate
thelytoky hypothesis is further supported by the absence of evidence indicating sexual reproduction or genetic
recombination across the species’ extensive distribution range (Mexico-Argentina). Potential conflicting evidence for sexual
reproduction in this species derives from three Mycocepurus males reported in the literature, previously regarded as possible
males of M. smithii. However, we show here that these specimens represent males of the congeneric species M. obsoletus,
and not males of M. smithii. Mycocepurus smithii is unique among ants and among eusocial Hymenoptera, in that males
seem to be completely absent and only queens (and not workers) produce diploid offspring via thelytoky. Because colonies
consisting only of females can be propagated consecutively in the laboratory, M. smithii could be an adequate study
organism a) to test hypotheses of the population-genetic advantages and disadvantages of asexual reproduction in a social
organism and b) inform kin conflict theory. For a Portuguese translation of the abstract, please see Abstract S1.
Citation: Rabeling C, Lino-Neto J, Cappellari SC, Dos-Santos IA, Mueller UG, et al. (2009) Thelytokous Parthenogenesis in the Fungus-Gardening Ant Mycocepurus
smithii (Hymenoptera: Formicidae). PLoS ONE 4(8): e6781. doi:10.1371/journal.pone.0006781
Editor: Corrie S. Moreau, Field Museum of Natural History, United States of America
Received June 6, 2009; Accepted July 7, 2009; Published August 26, 2009
Copyright: ! 2009 Rabeling et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: Funding was provided by an Ernst Mayr Travel Grant in Animal Systematics (Museum of Comparative Zoology), the Green Fund (Harvard University), a
NSF Doctoral Dissertation Improvement Grant (DEB-0808164), as well as research grants from the Section of Integrative Biology from the University of Texas at
Austin to CR; a Dorothea Bennett Memorial Graduate Fellowship to SCC; the Funding was provided by an Ernst Mayr Travel Grant in Animal Systematics (Museum
of Comparative Zoology), the Green Fund (Harvard University), a NSF Doctoral Dissertation Improvement Grant (DEB-0808164), as well as research grants from the
Section of Integrative Biology from the University of Texas at Austin to CR; a Dorothea Bennett Memorial Graduate Fellowship to SCC; the Fundação de Apoio à
Pesquisa do Estado de São Paulo (2006/00185-7) and the Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico (476250/2008-0 and 310826/2006-3) to
MB; the Conselho Nacional de Desenvolvimento Cientı́fico e Tecnológico to JLN and IAS; and the National Science Foundation (DEB-0639879) to UGM. The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: [email protected]
Introduction
Asexuality is part of normal hymenopteran reproduction
because males are produced via arrhenotoky (males develop from
unfertilized, haploid eggs) [15,16]. In contrast, females develop
from fertilized, diploid eggs [17,18]. Thelytokous parthenogenesis,
the production of diploid female offspring from unfertilized eggs,
has been observed in a small number of eusocial hymenopteran
species. So far, thelytoky has been convincingly demonstrated in
the Cape honeybee, Apis mellifera capensis [19–21], and for seven
distantly related species of ants (see Discussion).
Mycocepurus smithii is a common, but cryptic species of fungusgardening ants, which is widely distributed throughout Latin
America and obligately relies on a symbiotic fungus for food [22–
24]. Recently, independent lines of evidence indicated that M.
smithii, might be capable of thelytokous reproduction. First, the
absence of males in three years of continuous fieldwork in Puerto
Rico led Fernández-Marı́n et al. [25] to hypothesize that queens of
Mycocepurus smithii might reproduce via thelytoky. A comparative
Explaining the prevalence of sexual over asexual reproduction
has been a longstanding goal in evolutionary biology [1–5]. Over 20
models describing the advantages of sex have been proposed,
differing with respect to the short- and long-term advantages of
sexuality that outweigh its two-fold evolutionary cost [2,6–8]. The
most generally applicable models postulate fitness benefits for
recombining organisms, either because of faster adaptation to
changing conditions (e.g., the Red-Queen model) [9,10], or because
of more efficient purging of deleterious mutations (mutation-based
models) [11,12]. The near absence of asexual lineages that persist
over long evolutionary time suggests that the disadvantages of
asexuality generally outweigh its benefits [13,14]. However, asexual
organisms arise sporadically throughout the tree of life, and the
study of obligate long-term asexual organisms may hold the answer
to why almost all eukaryotes reproduce via meiosis and syngamy.
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study of Mycocepurus populations in Brazil found that in contrast to
M. goeldii, M. smithii nest entrances occurred in high densities and
that nests seemed to be connected to each other, forming a colony
network [26,27]. The connected nest systems of M. smithii
suggested that colonies multiply by fission, potentially influenced
by the reproductive biology of the species [26,27]. Additionally,
Himler et al. [28] observed thelytoky of M. smithii in laboratory
colonies from Panama. Males were not encountered in M. smithii
nests during the Mycocepurus mating season in Peru, Brazil
(Rabeling, pers. obs.), Puerto Rico [25] and Panama [28], even
though sympatric, sexually reproducing Mycocepurus species
engaged in nuptial flights, suggesting that M. smithii might be
obligately asexual.
The existence of three unidentified Mycocepurus males collected
by W. E. Kerr in Rio Claro, São Paulo State in Brazil [29] appear
to challenge the hypothesis that M. smithii is obligately asexual.
Until recently, only two Mycocepurus species were known from São
Paulo State: M. goeldii and M. smithii. The males of M. goeldii had
been previously described [30], and hence, by process of
elimination, the males collected by Kerr were tentatively assigned
to M. smithii. Interestingly, Kempf [22] described the morphology
of these males in his taxonomic revision of the genus, but had the
foresight to not name them, because the specimens were
unassociated with a maternal colony, making it impossible to
identify them with confidence. The ‘Kerr males’ therefore could
belong to an undescribed species or to a described species not yet
known from São Paulo State.
In order to test if thelytoky is widespread in queens of M. smithii
and to investigate the behavioral ecological consequences of
thelytoky in field populations, we studied the population biology,
reproductive physiology of queens and workers, and the mating
behavior of a M. smithii population in Brazil, and compared this
population with two sexually reproducing, sympatric congeners. In
addition, we provide taxonomic evidence for the identity of the
‘Kerr males’.
M. smithii nests do not occur in layers deeper that 70 cm (Rabeling,
unpublished). In order to excavate and survey all underground
chambers of the entire aggregation, a trench of 150 cm depth was
dug outside the marked study plot at a distance of at least 50 cm
from the first nest entrance. Vertical slivers of soil were then sliced
away with a sharpened spade. The position of all nest chambers
was recorded in a grid documenting the spatial distribution within
the study population. The digging was continued 50 cm past the
limits of the study area.
Colonies of Mycocepurus goeldii and an undescribed Mycocepurus
species served as a positive control for sexually reproducing
species. Nests of M. goeldii were shown to have a single nest
entrance [27]. Single, spatially separated colonies were chosen and
a trench of 250 cm depth was dug in 50 cm distance from the first
nest entrance, since pilot study showed that M. goeldii chambers
could be encountered as deep as 190 cm below the surface
(Rabeling, unpublished). Vertical slivers of soil were sliced away
with a spade and the position of all nest chambers were also
Materials and Methods
Population Biology of Mycocepurus smithii
Fieldwork was conducted from September 29th to October 26th
2008 on the Campus of São Paulo State University (UNESP) in
Rio Claro, Brazil (22.3955uS, 047.5424uW; elevation 608 m). In
the State of São Paulo, the nuptial flights of most attine ant species,
including those of Mycocepurus [29], occur during late September
and October, which mark the beginning of the rainy season. In
order to maximize the chance of finding males in the nests of M.
smithii, we focused collecting on the probable mating season.
Previous studies demonstrated that it might be difficult to
recognize M. smithii nests as separate units because nest entrances
may occur in high densities and lateral tunnels connect
underground chambers [27]. We therefore selected a study site
where nest entrances were spatially separated from other M. smithii
populations and an entire nest aggregation could be studied
through excavation, including all reproductively active and
potentially inseminated queens. Once the target aggregation was
identified, the study area was marked and the nest entrances
mapped, resulting in a 3.5 m62 m study area with 10 nest
entrances (Fig. 1). Throughout the excavation process, the study
plot was frequently sprayed with water to increase the visibility of
nest entrances (the ants open the nest entrances after the entrance
hole has been closed by washed-in soil). These freshly excavated
soil mounds are easier to recognize than older nest entrances,
which sometimes erode over time during periods of drought. A
pilot study conducted at the same site in 2006 demonstrated that
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Figure 1. Spatial distribution of nest entrances and fungus
chambers of the studied Mycocepurus smithii population. Nest
entrances are depicted as Xs, queenright chambers as solid black dots and
all other fungus chambers as open black circles; size of circles does not
represent the relative sizes of nest chambers. The x and y axes are labeled
in cm. The nest excavation was continued 50 cm beyond the study plot
limits in all four directions and no additional chambers were found.
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21, 42, 47 & 50; see Table S1) and dissected following the
methodology described above.
recorded in a grid. The digging was continued 50 cm past the last
excavated chamber.
All Mycocepurus specimens were stored in 95% ethanol for
further examination and censusing. Voucher specimens of this
study were deposited in the entomology collection of Maurı́cio
Bacci’s Molecular Evolution Laboratory at São Paulo State
University (UNESP), the Museum of Comparative Zoology at
Harvard University (MCZ), the Museu de Zoologia da USP
(MZUSP), and in C. Rabeling’s collection.
To supplement the results of the M. smithii nest excavations and
provide direct evidence for polydomy, different nest casting
methods were tried, following methodologies described by
Williams and Lofgren [31] and Tschinkel [32–34]. However, the
tunnel diameter of M. smithii nests was as narrow as 1 mm, which
did not permit plaster of Paris or molten zinc to trace the tunnels.
Other casting techniques will be required in the future to illustrate
that nests of M. smithii are connected via tunnels.
Behavioral observations of nuptial flights
Under thelytoky and in the absence of males, the mating
behavior is expected to degenerate, including the behavioral
preparations of workers when modifying nest entrances for mating
flights. Preliminary behavioral observations in previous years
showed that the sexually reproducing Mycocepurus goeldii modifies its
nest mounds in preparation of the nuptial flights of the alates (see
results for details). These modified nest entrances serve as a proxy
if a particular colony is preparing for a nuptial flight. Between
September 29th and October 15th the nest entrances of ten M.
goeldii colonies were monitored three times per day and compared
to the ten entrances of M. smithii in the study plot.
Taxonomic identity of the ‘Kerr males’
For most Mycocepurus species only the worker caste is described.
To find males that match the ‘Kerr males’, special emphasis was
given to collecting workers, queens, and males from any
Mycocepurus colonies. Mycocepurus obsoletus is one of the species
known only from workers and we collected entire colonies in a
Cerrado habitat at the margin of a gallery forest at the Reserva
Ecológica do IBGE, 35 km south of Brası́lia, in the Federal
District of Brazil (15.9436uS, 047.8767uW; elevation 1087 m). To
collect reproductives, the entrances of several nests were flagged
during the dry season in July 2008 and on November 12th 2008,
with the first rains of the season, alate Mycocepurus males (n = 19)
and queens (n = 13) were collected next to the previously marked
nests. The external morphology of the males was compared to the
‘Kerr males’ under a Leica Z6 APO dissecting scope.
Additionally, these males were identified to species using
molecular sequence information to ensure the males from Brası́lia
are in fact Mycocepurus obsoletus males. The partial DNA of three
single-copy nuclear markers (Elongation Factor 1-alpha F1 copy,
Wingless and Long Wavelength Rhodopsin) and one mitochondrial gene (Cytochrome Oxidase I) were sequenced, resulting in
3512 base pairs, and compared to sequence information from M.
obsoletus workers collected at the same site. The resulting molecular
phylogeny will be presented in the context of an ongoing generic
revision (C. Rabeling in prep.).
Reproductive Biology of Mycocepurus queens
All Mycocepurus specimens were collected in 95% ethanol and
dissected in 0.2 M phosphate buffer. Female reproductive tracts
were examined using an Olympus SZ40 dissecting scope and an
Olympus CX31 microscope. Photographs of the female reproductive tracts were taken with a Nikon D200 SLR digital camera
mounted on an Olympus SZH10 dissecting scope or an Olympus
CX31 microscope. To test if queens were inseminated, the
spermathecae were dissected, opened and examined under a
microscope, to ensure that the content were sperm and no other
kind of proteinaceous secretion. The developmental status of the
ovaries was recorded as undeveloped (0), developing without fully
developed oocytes (1) and fully developed ovaries with mature
oocytes present (2) (Table 1). The presence of yellow bodies
(corpora lutea) at the base of the ovaries was also recorded. Yellow
bodies are remnants of follicular cells and indicate if the respective
female oviposited in the past. In case of a parthenogenetic,
reproductively active female, one would expect an empty
spermatheca, fully developed ovaries with mature oocytes, and
corpora lutea as an indicator of oviposition activity in the past.
Reproductively inactive females could have both empty and
sperm-filled spermathecae, depending on insemination status, but
their ovaries are expected to be undeveloped with no yellow
bodies. In contrast, a sexually reproducing female would have a
sperm filled spermatheca, developed ovaries, and yellow bodies.
To test if queens were the only reproductively active individuals
in the colonies a total of 50 workers were dissected. Five workers
were randomly chosen from five queenright (chambers 4, 30, 37,
45 & 59; see Table S1) and five queenless chambers (chambers 13,
Results
Population Biology of Mycocepurus smithii
A total of ten M. smithii nest entrances were identified in the 7 m2
study plot (1.43 entrances/m2) (Fig. 1). The excavation of this
population revealed a total of 59 fungus chambers (see Table S1 for
Table 1. Reproductive status of dissected Mycocepurus queens.
species
n
spermatheca
ovary development (*)
corpora lutea
M. smithii (dealate)
12
translucent/empty
2
present
M. goeldii (dealate)
2
opaque w/sperm
2
present
M. goeldii (alate)
5
translucent/empty
0
absent
M. sp. nov. (dealate)
15
opaque w/sperm
1 (n = 12); 2 (n = 3)
absent (n = 12); present (n = 3)
M. sp. nov. (alate)
2
translucent/empty
0
absent
(*) The state of ovary development was divided in three categories: 0 = not developed, 1 = developing but without fully developed eggs, 2 = well developed with mature
eggs present. In nests of M. sp. nov. more than one queen was found in a single chamber. Among those queens some were reproductively active, while others were
inseminated, but the ovaries were still developing. The presence of corpora lutea indicates if a female oviposited.
doi:10.1371/journal.pone.0006781.t001
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ovarioles, which did not contain differentiated premature oocytes,
and yellow bodies were absent. The queens of M. sp. nov. could be
divided in three reproductive categories. First, dealate, reproductively active queens (n = 3) had sperm-filled spermathecae,
developed ovaries containing mature oocytes and corpora lutea.
Second, dealate, inseminated queens (n = 12), which did not have
fully developed ovaries and lacked yellow bodies. Instead the
ovarioles of each ovary were in the process of development, and
did not contain mature oocytes, but premature oocytes were
present. Third, alate, virgin queens (n = 2), which had empty
spermathecae, undeveloped ovaries and absent corpora lutea.
The examined queens of M. goeldii and M. sp. nov. confirmed
the predictions for sexually reproducing species: virgin, non
inseminated queens had undeveloped ovaries and the absence of
corpora lutea indicated that the respective female never oviposited.
In contrast, the reproductively active females had sperm-filled
spermathecae, developed ovaries and the presence of corpora lutea
indicated oviposition. Some dealate queens, which were not
reproductively active (indicated by the absence of corpora lutea),
were also inseminated and had developing ovaries.
None of the 50 examined M. smithii workers was reproductively
active. Each worker had a pair of ovaries with a single ovariole per
ovary. Ovarioles did not contain mature oocytes and yellow bodies
were absent. All workers were lacking a spermatheca.
exact chamber contents). The entire population consisted of 851
workers and 12 queens, averaging approximately 14 workers per
chamber and one queen in every 5th fungus chamber (see Table S1).
The maximum number of workers per chamber was 43, the
minimum one. All queenright chambers contained only a single
reproductively active queen, however, the number of queens
encountered was higher than the number of nest entrances in the
study area. The brood was distributed in 21 (36%) chambers and
consisted of 115 early instar larvae, 40 worker pupae, 51 queen
larvae and 274 queen pupae. The sexual brood was restricted to ten
chambers (17%) and in eight of them worker brood and sexual
brood co-occurred. The maximum and minimum number of sexual
brood per chamber was 80 and one, respectively. No male pupae
and no alate queens and males were present in the nest chambers.
Because of the close proximity of some of the nest entrances
(Fig. 1), it was not possible to assign some chambers to a single nest
entrance. However, the chambers were distributed in clusters close
to the nest entrances. Nest entrance #1 was located 116 cm
distant to the next entrance (#3) with 15 fungus chambers
underneath, four of them queenright. Under entrances 2, 3 and 4
a total of 15 chambers was encountered, three of them queenright.
Six fungus chambers were located under entrances 5 and 6, of
which one contained a queen and entrance number 7 possibly led
to nine fungus chambers, two of them containing a reproductively
active queen. Twelve fungus chambers were associated with
entrances 8, 9 and 10, of which a single chamber contained a
queen. Two chambers, one of them queenright (Fig. 1), were
almost equidistant to entrances 4, 5/6 and 7 and no entrance was
encountered above these chambers.
The spatial distribution of queenright chambers and nest
entrances were not paired, indicating that some of the colonies
have more than one reproductively active queen, while others use
more than one entrance to access their nest. The fungus chambers
were distributed in depths between 10 and 66 cm below the
surface, well above the depth of the excavation pit of 150 cm
depth throughout the study plot.
The spatial distribution of M. goeldii entrances and queenright
chambers were exactly paired. Each of the two nests excavated
had a single nest entrance, and each nest was recognizable as
single spatially separate units, containing five or six fungus
chambers, respectively. Both colonies had a single reproductively
active queen.
Behavioral observations of nuptial flights
Nest observations started on September 29th in Rio Claro, before
the onset of the rainy season. At that time, nest entrances of M. goeldii
and M. smithii consisted of loose, irregularly shaped soil mounds of 5–
20 cm diameter, each with a single entrance. Rains started on October
2nd, stimulating M. goeldii workers to increase the number of nest
entrances to about 20–30 entrances per nest mound. Consequently, the
soil mound surrounding the entrances had a sponge like appearance.
Several M. goeldii workers waving their antennae surrounded each
entrance hole. Upon modification of the mound, males of M. goeldii
could be observed moving in the passages of the sponge like nest
mound, although the males did not surface. Queens were not observed.
On October 3rd, cooler weather and rain showers caused temperatures
to drop by 5–10uC. During this period, the nest entrances of M. goeldii
stayed open or were reopened after rain had washed soil into the
entrance tunnels. The workers of M. goeldii were still surrounding the
entrance holes and alate males and queens could be seen in the nest
mound tunnels. On the morning of October 7th, the temperature had
increased to about 25uC, and the winds and the rain had stopped. At
approximately 10.00 h, the males of M. goeldii colonies started the
nuptial flight. First resting on the rim of each entrance crater, then
spreading the wings and flying up vertically, perhaps gathering in the
lower branches of a tree, as observed by Kerr [29]. The alate queens
started flying slightly later at 11.30 h. The release of alates from each
colony continued until 15.00 h. During the rest of the afternoon,
workers closed the supernumerary nest entrances, until each colony
had only a single entrance.
During this same time period, and until the end of the study
three weeks later, the entrances of M. smithii nests remained
unmodified, with some soil surrounding a single entrance hole. In
addition, neither mating flights, nor modified nest mounds, nor
males were ever observed throughout several years of field
research in Latin America (Rabeling, field observations 2003–
2008).
Reproductive Biology of Mycocepurus queens
Since spermatheca and ovary morphology are variable among
different ant genera, we dissected the reproductive tracts of 36
Mycocepurus queens (M. smithii, n = 12; M. goeldii, n = 7; M. sp. nov.,
n = 17; see Table 1). Dealate and alate queens of M. goeldii and an
undescribed Mycocepurus species served as positive controls for
inseminated queens and as negative controls for non-inseminated
females, respectively. Twelve queens of M. smithii had translucent
spermathecae, not revealing any content upon opening. The
ovaries of these females were well developed and contained several
mature oocytes per individual. The base of each ovariole had
multiple corpora lutea, indicating the reproductive activity of each
female (Fig. 2). Hence, all M. smithii queens showed the signs of
thelytokous reproduction: empty spermathecae, developed ovaries,
and presence of corpora lutea confirming past oviposition. In
contrast, the two dealate M. goeldii queens both had opaque
spermathecae with sperm content. The ovaries of both queens
were well developed with several mature oocytes present and the
base of each ovariole contained multiple corpora lutea, confirming
reproductive activity of these females. Five alate, virgin M. goeldii
queens had empty spermathecae, undeveloped ovaries with short
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Taxonomic identity of the ‘Kerr males’
On November 12th, 19 males and 13 queens were collected
when exiting the marked colonies of Mycocepurus obsoletus. The
previous day, the first heavy rainstorm of the season soaked the soil
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Figure 2. Internal reproductive organs of Mycocepurus smithii (A, B) and M. goeldii queens (C, D) in reflecting light (A, C) and
transmission light (B, D). Arrows drawn from the top left to the bottom right point to the spermatheca and arrows drawn from the top right to the
bottom left point to yellow bodies. (A, B) The spermatheca of M. smithii is translucent (empty), the ovaries are developed and yellow bodies are
present. (C, D) A reproductively active M. goeldii queen has an opaque, sperm-filled spermatheca, developed ovaries and yellow bodies. The scale bar
represents 2 mm in all images.
doi:10.1371/journal.pone.0006781.g002
appear to be specimens of M. obsoletus. A comparison of the male
sequences to sequences of M. obsoletus workers from the same
locality resulted in 100% sequence identity, confirming the identity
of those males. Consequently, the ‘Kerr males’ are the males of M.
obsoletus and not the males of M. smithii.
for approximately 24 hours and stopped at 10.00 h of November
12th. Two hours later (12.00 h) alate males and queens were
observed to lift off from the leaf litter for 10–50 cm before landing
again in the litter. The nuptial flight lasted for approximately two
hours from noon to 14.00 h and was interrupted by a brief rain
shower; after the rain shower (15.00 h), no activity could be
detected at the marked nests. It is unclear whether alates flew into
the surrounding vegetation and formed mating aggregations, as
has been reported for M. goeldii [29], or mated in the leaf litter.
The morphological comparison of the alate males to the males
collected by Kerr in 1960 revealed that males from both
collections, Brası́lia and Rio Claro, are almost identical (Fig. 3).
Identifying characters for Mycocepurus males include the shape of
the propodeal spine, the petiole and the wing venation (Fig. 3).
Given the morphological variability observed within and between
all species of Mycocepurus (Rabeling in prep), the ‘Kerr males’
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Discussion
Several lines of direct evidence (queen reproductive physiology)
and indirect evidence (taxonomy & natural history) implicate
obligate thelytokous parthenogenesis for a Brazilian population of
the fungus-gardening ant species M. smithii. The presence of
mature oocytes and yellow bodies indicate that dissected queens
had oviposited and were reproductively active, whereas the empty
spermathecae prove that queens were not inseminated. In
combination, these characteristics indicate thelytokous partheno5
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Figure 3. Morphological comparison of one of the Mycocepurus males collected by W. Kerr in Rio Claro in 1960 (A, B, C) and a
Mycocepurus obsoletus male (D, E, F). (A) lateral view of ‘Kerr male’, (B) forewing and (C) hindwing in dorsal view. (D) Male of Mycocepurus
obsoletus in lateral view, (E) forewing and (F) hindwing in dorsal view. Morphological characters informative for species identification are the shape of
the propodeal spine, of the petiole and the wing venation. Scale bars represents 1 mm in all images.
doi:10.1371/journal.pone.0006781.g003
genetic propagation and support Fernández-Marı́n’s [25] hypothesis of asexuality in M. smithii, formulated to explain the absence of
males in a Puerto Rican M. smithii population.
Three males from Rio Claro in Brazil [22,29] represented a
challenge to the obligate asexuality hypothesis, because they were
suspected to be males of M. smithii, indicating the potential for sex
and recombination in this species. Our morphological study,
however, indicates that the ‘Kerr males’ are the previously unknown
males of M. obsoletus, not males of M. smithii. Previously, M. obsoletus
was not known to occur in Rio Claro, but we now know that M.
obsoletus has a Brazilian Cerrado distribution (Fig. 4), thus its
occurrence in the Rio Claro area is certainly possible. In addition,
M. obsoletus workers have been collected at two other relatively
nearby localities in São Paulo State (Fig. 4; see also [35]), further
increasing the likelihood of its occurrence at or near Rio Claro.
Thus the ‘Kerr males’ seem to represent only the southernmost
presently known distribution record of M. obsoletus (Fig. 4).
Matching the ‘Kerr males’ to M. obsoletus provides only
circumstantial evidence for obligate thelytoky, but it does remove
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the strongest potential counter evidence, i.e.: that the ‘Kerr males’
are actual males of M. smithii. While we cannot rule out the
possibility that M. obsoletus and M. smithii males are morphologically identical; this is most unlikely, however, given the observed
morphological variation within and between Mycocepurus species
(Rabeling, pers. obs.).
Within eusocial Hymenoptera, arrhenotokous parthenogenesis
is a widely distributed phenomenon [15,36] and the reproductive
modes in ants are particularly diverse [37]. In contrast, thelytokous
parthenogenesis, the production of female offspring from unfertilized eggs, has been rarely confirmed in eusocial Hymenoptera,
although the number of recognized thelytokous species is
increasing. So far, thelytoky has been demonstrated convincingly
for the South African honey bee subspecies Apis mellifera capensis
[19–21] and seven ant species, which can be divided into three
categories of asexual reproduction: queens produce queens and
workers via thelytoky, but workers are sterile and males seem to be
absent (called here: type A thelytoky); workers produce workers
and potentially queens via thelytoky (type B); or queens produce
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Asexual Fungus-Gardening Ant
Claro, Brazil population are thelytokous. Himler et al. [28]
dissected six queens from a Panamanian population, reaching the
same conclusion. Based on these three geographic data points,
Brazil, Panama, both confirmed, and Puerto Rico, still unconfirmed, it seems that thelytoky is widespread across M. smithii’s
distribution range.
Firm evidence capable of identifying the cytological mechanism
of thelytoky (i.e., apomixis versus automixis) in M. smithii is
currently lacking. A comprehensive genetic population study in
combination with cytological evidence is necessary to distinguish
between meiotic and mitotic parthenogenesis. Himler et al. [28]
tested queens and workers of 12 Panamanian colonies for allelic
variation at a single variable genetic locus and found queens and
offspring to be genetically identical, concluding that M. smithii
propagates clonally. However, during automixis with central
fusion, two haploid nuclei are produced during the first meiotic
division, which subsequently fuse to form the diploid zygote. Gene
recombination does not necessarily occur and thus, the genetic
signature of apomicticly and automicticly produced offspring can
be very similar if not identical [15,56,62,63]. A single genetic locus
is hence insufficient to distinguish between mitotic and meiotic
parthenogenesis.
Wolbachia bacteria induce parthenogenetic reproduction in some
parasitoid wasps [64]. Several hymenopteran species have been
tested for Wolbachia infections, but Wolbachia could never be
demonstrated to induce parthenogenesis in any thelytokous
eusocial Hymenoptera species [28,41,52,65]. This supports the
hypothesis that a sex determining system based on heterozygosity
may form a limitation to Wolbachia-induced parthenogenesis [65].
Wolbachia endosymbionts induce the production of homozygous
diploid offspring through gamete duplication [66,67], which
results in the exclusive production of diploid males. In ants and
eusocial bees, females are required to be heterozygous at the sexdetermining locus, a condition difficult to achieve when parasitized
by a gamete duplicating endosymbiont [21,65]. Because thelytoky
seems to have evolved independently of Wolbachia in ants and bees,
it also seems unlikely that thelytoky of M. smithii queens was
induced by Wolbachia. The absence of Wolbachia in M. smithii
supports this expectation and additional antibiotic treatment could
not ‘cure’ M. smithii from parthenogenesis [28].
The wide geographic distribution could suggest an ancient
origin of asexuality in M. smithii. Low variability in DNA sequence,
however, does not support an ancient asexuality hypothesis, but
rather argues for a recent origin of thelytoky followed by a rapid
population expansion (Rabeling, unpublished). The widespread
geographic distribution also suggests that geographic parthenogenesis, the occurrence of parthenogenetically reproducing
lineages towards the margin of a distribution range [68,69],
unlikely applies to M. smithii, because both São Paulo State and
Panama are located well within the continuous distribution range,
which extends from Northern Mexico to Argentina [22,23;Rabeling, in prep].
Theory predicts that morphological structures degenerate if not
maintained by natural selection. Panamanian M. smithii queens
apparently lack the so-called mussel organ, and Himler et al. [28]
therefore argued for degenerate mating apparatus, which is
thought to support an ancient origin of asexuality in M. smithii.
The mussel organ is a sclerotized structure in the reproductive
tract of leaf cutter ant queens, which is thought to protect the
female genitalia tissue from damage inflicted by sclerotized hooks
of the male’s penis [70]. In an obligately asexual species both the
spermatheca and the mussel organ are expected to lose their
function. Given that soft tissues like the spermatheca were present
and not reduced in size in M. smithii queens when compared to two
Figure 4. Geographic distribution of Mycocepurus obsoletus.
Thick black lines depict the boundaries of Brazilian States, open circles
represent collections based on worker specimens, the solid black dot
depicts the collection site of the ‘Kerr males’: Rio Claro in São Paulo
Sate. From north to south, M. obsoletus has been recorded in the States
of Pará, Tocantins, Goiás, the Federal District, Minas Gerais and São
Paulo. Besides Rio Claro, M. obsoletus has been collected in two other
localities in São Paulo Sate: a) Reserva Ecológica de Jataı́ near Luis
Antônio (23–25.V.1997, Silvestre & Silva col.; see [35]), which is located
approximately 100 km north of Rio Claro, and b) Ibitinga (25.I.1964, K.
Lenko col.), which is approximately 150 km northwest of Rio Claro.
doi:10.1371/journal.pone.0006781.g004
queens thelytokously and workers sexually, and workers produce
queens thelytokously (type C). Males are known for all type B and
type C asexual ant lineages, suggesting the potential for sex and
recombination, but males remain unknown for type A asexuals, of
which M. smithii is the first known example in the Formicidae.
Among asexual ant species, worker thelytoky (type B thelytoky)
occurs in four distantly related ant species, the cerapachyine ant
Cerapachys biroi [38–40], the two myrmicine ants Messor capitatus
[41] and Pristomyrmex punctatus [42–48], and the ponerine ant
Platythyrea punctata [49–52]. The thelytokous production of queens
by queens and sexual production of workers by queens (type C)
has so far been documented in the formicine ant Cataglyphis cursor
[53–56], and the two myrmicines Wasmannia auropunctata [57–59]
and Vollenhovia emeryi [60–61].
At present, Mycocepurus smithii seems to be the only representative of type A asexual ant species. Natural history studies in Puerto
Rico [25] and the Brazilian Amazon [26,27] suggested an unusual
reproductive biology for M. smithii. Fernández-Marı́n et al. [25]
first proposed the hypothesis that M. smithii might reproduce
thelytokously. This study shows that M. smithii queens from the Rio
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Asexual Fungus-Gardening Ant
cultivars, which moreover spanned almost the entire diversity of
lower attine cultivars [26,79]. No firm conclusion can be drawn
from these preliminary results, but the observed pattern is
consistent with the hypothesis that asexuals exploit new food
sources effectively and colonize new habitats rapidly. A similar
effect can be observed in cyclical parthenogenetic animals like
water fleas (Daphnia), aphids, flukes or some gall wasps living in
seasonal environments: during the summer, food sources are
abundant and reproduction switches to parthenogenesis, whereas
later in the season, when resources become scarce, populations
adopt sexual reproduction [4, pp. 57–62]. Because M. smithii
inhabits both tropical and subtropical habitats, the need for sex to
persist in seasonal environments may not be pressing, but a
comprehensive comparison of Mycocepurus cultivars grown by
asexual and sexual hosts will have to test this hypothesis more
thoroughly.
Several naturalists have discussed the possibility that M. smithii
colonies are polydomous, i.e., single colonies occupy more than
one nest [22,25,27,83,84]. Conflicting reports stem from frequent
observations of the patchy distribution and high nest densities
characteristic of M. smithii populations. Small areas often contain
multiple nest entrances, as much as six entrances per square meter
[26], whereas adjacent areas may have none at all. So far, only two
studies have investigated the nest architecture systematically.
Fernández-Marı́n et al. [25] concluded that M. smithii colonies had
a single nest entrance in Puerto Rico, whereas Rabeling et al. [27]
found that in the Brazilian Amazon large nest systems were
interconnected through lateral tunnels, potentially enabling the
exchange of workers and brood, which would argue for polydomy.
In the Rio Claro M. smithii population, some nest chamber
clustered underneath 2 or 3 spatially separated nest entrances (6
chambers below entrances #5 and #6 and 12 chambers below
entrances #8, #9 and #10; Fig. 1) and cluster contained only a
single reproductively active queen. In addition, a queenright
chamber was located in equal distance to four nest entrances (#4,
#5/6 and #7; Fig. 1) and this chamber had no detectable
entrance above it. These patterns suggest that nests are connected
underground and workers move freely between chambers, sharing
the same entrances, further supporting polydomy. The alternative
hypothesis could be that additional, potentially inseminated
queens of the population escaped during excavation and that
some entrances remained inactive and thus remained undetected
during the study period of a month. Because of high levels of
relatedness are expected for a strictly thelytokous species, it might
be that M. smithii might not only be polydomous but also
unicolonial.
Eidmann [85] first hypothesized that M. smithii colonies were
polygynous, because ‘‘in every of the numerous examined fungus
chambers’’ he found ‘‘several old, dealate queens’’ (p. 86).
Unfortunately, Eidmann never tested his hypothesis by dissecting
the queens. Fernández-Marı́n et al. [25] further substantiated
Eidmann’s hypothesis, suggesting that M. smithii colonies might be
secondarily polygynous, because pleometrosis, the foundation of
colonies by multiple queens, does occur in some colonies.
Dissections confirmed that multiple queens from the same nest
were reproductively active, as indicated by the presence of
developed ovaries and yellow bodies. However, it remains unclear
if the dissected queens were found in the same fungus chamber or
if they could have been reproductive females from different
colonies. In addition, Amazonian M. smithii colonies had several
dealate queens in the same fungus chamber, but their reproductive
status was not confirmed through dissections [27].
In the Rio Claro population, only a single queen was found per
fungus chamber (n = 10). The presence of several queenright
sexual congeners, it seems premature to argue for ancient
asexuality based on the absence (or non-detection) of the mussel
organ. Ideally, a comparative functional-morphological study
should first establish a) whether the mussel organ is present in
closely related, sexually reproducing Mycocepurus species, because
Mycocepurus and Atta shared the most recent common ancestor
some 50 Million years ago and are therefore only distantly related
to each other [71]; b) whether the mussel organ in M. smithii is
homologous to the respective structure in A. colombica for which this
organ was first described [70]; and c) to what degree sclerotization
varies with age, because weakly sclerotized structures are often
translucent in recently eclosed arthropods.
High relatedness is expected in colonies of M. smithii whether
queens reproduce via apomictic or automictic thelytoky. Kin
conflict theory predicts conflict between individual kin or groups of
kin (i.e., castes) within a colony, because individuals or castes differ
in relatedness, reproductive success or both [72]. In a society of
highly related or even clonal organisms kin conflict over
reproduction is expected to be reduced if not entirely absent.
However, Hamilton’s rule [73,74] does not account for low
frequency mutations with strong selective effects and therefore,
relatedness alone is not always a good predictor of kin conflict over
reproduction [75,76]. Once the genetic underpinnings of reproduction in M. smithii societies are understood, this ant should be an
interesting study organism for kin selection theory.
Behaviors specific to sexual reproduction might degenerate
rapidly in parthenogenetic lineages [77]. In M. smithii, modifications of the nest related to nuptial flight activity were never
observed during six field-seasons (2003–2008), even though alate
queens were present in M. smithii nests. At the same time sympatric
sexual species showed modified nest structures and performed
nuptial flights. The observation that M. smithii queens were still in
the pupal stage whereas sexually reproducing species performed
nuptial flights suggests that M. smithii queens may be produced
over a wider time interval than in sympatric, sexual congeners. In
the absence of males, it is unnecessary for M. smithii queens to
depart in a synchronized population wide nuptial flight.
Consequently, it appears unnecessary to produce young queens
in synchrony and to open the nest entrance for mating flights,
which could possibly increase the risk of predation and parasitism.
Alternative to a nuptial flight, young queens could establish
themselves in a new chamber that is part of the maternal colony.
Such colony expansion would be consistent with the observation
that multiple reproductively active M. smithii queens inhabit the
same colony [25,27]. However, the ability of independent nest
foundation seems to persist in M. smithii, as infrequently alate M.
smithii queens can be found in the leaf litter (Rabeling, pers. obs.)
and Fernández-Marı́n et al. [25,78] observed claustral nest
founding of single dealate queens in Puerto Rican M. smithii.
Asexual lineages are capable of colonizing new habitats and
exploiting new food sources more efficiently than sexually
reproducing relatives [2,4,5]. Interestingly, M. smithii relies on
multiple, genetically distinct lineages of fungi for food [26,79,80].
Surveys of fungal diversity cultivated by attine ants suggested that
the majority of ants are associated with one fungal lineage [79] and
in the case of Cyphomyrmex ants, the existence of two distinct fungal
cultivar clades revealed the presence of two cryptic ant species
[81]. However, this trend does not necessarily apply to all species,
as was demonstrated by a comprehensive survey of microbial
diversity associated with the North American attine species,
Trachymyrmex septentrionalis [82]. This study identified four genetically distinct fungal lineages or species in 74 T. septentrionalis nests.
For M. smithii, fewer samples were screened (n = 20), but these few
samples already revealed twice the number of genetically distinct
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Asexual Fungus-Gardening Ant
chambers in one fungus chamber cluster below a single entrance
suggests that one polydomous colony can contain multiple
reproductively active queens, supporting functional polygyny.
Other clusters of fungus chamber, however, inhabited only a single
reproductively active queen. In case these clusters are not part of
the same polydomous nest, the Rio Claro M. smithii population
would comprise a mosaic of monogynous and polygynous colonies,
which would be consistent with Fernández-Marı́n et al. ’s [25]
hypothesis of secondary polygyny. Again, a genetic population
study will have to test if queens and worker offspring share the
same genetic signature or if multiple mothers produced the
workers of a single colony.
coupled with population genetic and phylogenetic studies will
certainly answer some of these questions and further increase the
already broad spectrum of reproductive diversity in eusocial
Hymenoptera and beyond.
Supporting Information
Abstract S1
Found at: doi:10.1371/journal.pone.0006781.s001 (0.03 MB
DOC)
Table S1
Found at: doi:10.1371/journal.pone.0006781.s002 (0.16 MB
DOC)
Conclusions
Three types of thelytokous reproduction have been documented
in eight distantly related ant species. Mycocepurus smithii appears to
be obligately asexual based on its reproductive physiology, and the
absence of males and mating behavior. A population genetic study
of M. smithii should show if this fungus-gardening ant species relies
exclusively on thelytoky or if cryptic sex does occur. Wolbachia
endosymbionts seem unlikely as parthenogenesis-inducing agent in
eusocial Hymenoptera in general. Ancient asexuality might be
suggested by the wide geographic distribution, but rendered
unlikely by presence of fully developed soft tissue (i.e., spermatheca) in the mating apparatus of M. smithii queens and low genetic
variability between populations. For all thelytokous ant species we
lack the kind of evolutionary ecology framework that might
indicate how long asexuality has persisted in the respective species
and what factors might have influenced the rise of thelytokous
populations from a sexually reproducing ancestor. The fact that
M. smithii cultivates multiple lineages of fungi is consistent with the
hypothesis that asexuals are efficient colonizers of novel habitats
and food sources. Due to the expected high relatedness within M.
smithii colonies this species is likely to inform kin conflict theory.
Careful natural history observations and behavioral experiments
Acknowledgments
We thank the Conselho Nacional de Desenvolvimento Cientı́fico e
Tecnológico (CNPq) and the Instituto Brasileiro do Meio Ambiente e
dos Recursos Naturais Renováveis (IBAMA) for permission to conduct
fieldwork in Brazil. We are grateful to Beto Brandão (MZSP), Stefan Cover
(MCZ), Ted Schultz (NMNH) and Heraldo Vasconcelos (UFU) for
contributing specimens to our study. We also thank the staff of the Reserva
Ecológica do IBGE for support during fieldwork. We would like to thank
Natalia Biani, Stefan Cover, Barrett Klein, Sasha Mikheyev, Jon Seal,
Kazuki Tsuji, and Phil Ward for commenting on earlier versions of this
manuscript, and four anonymous reviewers for constructive criticism. This
study is a chapter of Christian Rabeling’s doctoral dissertation at the
University of Texas at Austin.
Author Contributions
Conceived and designed the experiments: CR. Performed the experiments:
CR JLN IADS. Analyzed the data: CR. Contributed reagents/materials/
analysis tools: CR JLN SCC UGM MBJ. Wrote the paper: CR. Performed
fieldwork: CR SCC. Critically revised the manuscript: CR JLN SCC IADS
UGM MB.
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August 2009 | Volume 4 | Issue 8 | e6781